Construction and purification of site-specifically modified DNA templates for transcription assays.

Abstract

Chemical and physical agents can alter the structure of DNA by modifying the bases and the phosphate-sugar backbone, consequently compromising both replication and transcription. During transcription elongation, RNA polymerase complexes can stall at a damaged site in DNA and mark the lesion for repair by a subset of proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Although our understanding of DNA lesion effects on transcription elongation and the associated effects of stalled transcription complexes on DNA repair is broadening, the attainment of critical data is somewhat impeded by labor-intensive, time- consuming processes that are required to prepare damaged DNA templates. Here, we describe an approach for building linear DNA templates that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II. The method is rapid, making use of biotin-avidin interactions and paramagnetic particles to purify the final product. Data are supplied demonstrating that these templates support transcription, and we emphasize the potential versatility of the protocol and compare it with other published methods.